Apoptosis is an active form of cell death that is involved in multiple processes of normal cell development as well as in malignant cell transformations. Mechanism of apoptosis is engaged in biological events induced by various types of drugs, cytokines, and growth factors, oxidative stress, radiation, aging, autoimmune diseases, and immune rejection within organ transplantation. Recent studies on apoptosis demonstrate that common molecular mechanisms are employed in various types of apoptosis, induced by hormones, cytokines, growth factor deprivation, chemotherapeutic agents, ionizing radiation, immunological disorders, AIDS, cancer and aging (Nagata, (1997) Cell 88, 355-365).
Cascade-like activation of caspase proteases represents the fundamental point in the induction of apoptosis. Two distinct types of apoptosis signaling are described. The initial phase of receptor-depending triggering of apoptosis includes activation of appropriate death receptors by specific ligands, such as TNF or FasL, which are presently the most studied inductors of apoptosis . Upon activation, cell surface death receptors, Fas (CD95) or TNFR1, are attached to cytosolic adapter proteins (FADD, MORT, RIP, TRADD), which in turn recruit caspase-8 to activate the interleukin-1-β-converting enzyme ICE/CED-3 family protease (caspase) cascade, followed by activation of CPP32/caspase-3-subfamily of cysteine proteases, whose members occur in cell cytoplasm in the form of latent precursors, procaspases . Receptor-independent types of apoptosis usually include critically important cytochrome c-inducible mechanism that requires the formation of tertiary complex of cytochrome c, dATP, Apaf-1 and procaspase-9, that lead to the activation of the latter via autoproteolysis and homodimerization, and subsequent caspase cascade activation (Cohen et al. 1997 Biochem. J. 326: 1-16.).
Agents that affect the biological control of apoptosis thus have a potential therapeutic utility in numerous clinical applications. A variety of plant-derived inhibitors of apoptosis are employed to screen pathological disorders that often accompany chemotherapy, radiation, immune disorders, or AIDS. These supplements generally contain carbohydrates, fat and plant protein hydrolysates, lectins, and phospholipides (U.S. Pat. No. 6,004,579, Barr et al.). Potent regulators of apoptosis could be employed in the treatment of cancer patients to control cytokine therapy, chemotherapy, or radiation therapy. Apoptotic mechanisms operate at the various types of immunological disorders, such as autoimmune malignancies, immune rejection in organ transplantation or anaphylaxis, or viral infections with human immunodeficiency virus.
Alpha-fetoprotein (AFP) is a tumor-associated fetal glycoprotein displaying a wide range of biological activities, including cell growth regulation, differentiation of immature cells, immunosuppression of activated immune cells, tumor-specific induction of apoptosis and regulation of apoptotic signals mediated by other factors, as well as regulation of various gene expressions (Mizejewski, (2001). Exp. Biol. Med., 226: 377-408). Multiple evidences of cell growth regulative activities, including tumor suppressive activity, have been reported for various species of full-length AFP molecule , its proteolytic fragments or recombinant domains and synthetic peptides (Dudich et al. 1999, Biochemistry 38: 10406-104141; MacColl et al. 2001 Biochim. Biophys. Acta, 1528: 127-134). The search for the localization of functional active sites of the AFP molecule which are responsible for its multiple activities has been undertaken by various researchers. Localization of the arachidonic acid and estradiol binding sites have been successful (reviewed at Mizejewski, (2001). Exp. Biol. Med., 226: 377-408).
It was demonstrated recently, that AFP realizes its tumor-suppressive activity by triggering apoptosis through activation of caspase-3 and independently on Fas/FasL and TNF/TNFR signaling (Dudich et al., (1999) Eur. J Biochem. 266: 1-13; Semenkova et al., (1997) Tumor Biology, 18: 261-274; Dudich et al. (1998) Tumor Biology, 19:30-40). Multiple evidences of the AFP-mediated tumor cell growth suppression have been reported at the last decade, but the active site of the AFP molecule that is responsible for apoptosis signaling has not been identified. DNA and amino acid sequences of human AFP have been reported (Morinaga, et.al., “Primary structures of human alpha-fetoprotein and its mRNA” Proc. Natl. Acad. Sci. USA, 80:4604-4608 (1983). Synthetic peptides, corresponding to the E2-binding site were shown to possess tumor-suppressive activity (U.S. Pat. No. 5,674,842; October 1997 Mizejewsky; U.S. Pat. No. 5,707,963, July 1998 Mizejewsky). Variety of biologically active proteins shares a sequence homology with AFP (Mizejewski, (2001) Exp. Biol. Med. 226: 377-408). There has been identified authentic homology of AFP with various proteins, involved in apoptosis signaling, such as Bcl2, TNFR1, Fas, etc. (Mizejewski, (2001). WO9835981 A1 (Economou, J. et al. 1998) describes using of 66 AFP peptide sequences to be useful for immunization against cancer. One of the peptides, A20, was CRGDVLDCL, which incidentally happens to include a part of the active site of AFP, found in the present disclosure. However, no special effects by the peptide CRGDVLDCL were found and the present invention remains to be the first one able to identify one of the putative biologically active sites of AFP. Moreover, the peptides of the present invention include an additional cysteine residue which enables the formation of inter-chain disulphide bonds and to produce a significantly higher biological activity than the sequence CRGDVLDCL.
An important integrin binding site is a tripeptide Arg-Gly-Asp, which is present in a variety of intergrin ligands. Integrins are hetorodimeric glycoproteins mediating cell-matrix and cell-cell interactions and have an active role in the processes of cell differentiation, immune recognition, tumor development and metastatic growth. Contact regions for the Arg-Gly-Asp sequence have been identified in the integrin subunits (see Pasqualini, et al. J. Cell Biol. (1995)130: 1189-1196). Synthetic peptides containing the Arg-Gly-Asp motif are used as inhibitors of integrin-ligand interactions. It has been reported that synthetic peptides containing Arg-Gly-Asp motif are able of direct caspase-3 activation (Bukley, et al. (1999) Nature, 397: 534-539). AFP contains Arg-Gly-Asp (RGD) sequence in its sequence that it localized in the domain II in the position 253-255. According to the present invention, the RGD sequence is a part of the functionally active site of AFP involved in the apoptosis signaling.
The present invention describes the minimal part of the AFP molecule that is responsible for the apoptosis signaling. Small peptides or other small haptens are important regulators to be used as the drugs because they can be synthesized in vitro and they do not produce neutralizing antibodies. Peptides containing from 6 to 10 residues were produced with the aid of the F-MOC solid phase chemistry. The similar peptides corresponding to homologous sequences of human serum albumin (HSA) were also synthesized. All peptides were assessed for their growth regulative activity in whole cells in culture and also tested for their ability to directly induce caspase activation in cell-free cytosolic extracts. Additionally, the peptides were assessed for their ability to modulate apoptosis induced by AFP and anti-Fas cytotoxic Mabs CH-11 in whole cells and to affect cytochrome c-induced caspase activation in cell-free systems.